Mite galectin

ABSTRACT

A novel galectin, a polynucleotide encoding the same, a vector and a transformant comprising the polynucleotide, an antibody against the galectin, and a screening method for screening a substance capable of modifying the galectin, are disclosed. According to the galectin, polynucleotide, or vector, it is possible, for example, to exterminate ticks, or to treat or prevent tick-borne infections such as rickettsiosis, filariasis, Q fever, African recurrent fever, or viral encephalitis.

TECHNICAL FIELD

This invention relates to a tick galectin.

BACKGROUND ART

Ticks are the cause, directly or indirectly, of extensive damage toanimals or humans. Examples of the direct damage are pruritus orbleeding caused by biting or blood-sucking, or tick paralysis orallergic diseases caused by saliva secreted in blood-sucking orregurgitation of midgut contents. Examples of the indirect damage arevarious diseases in livestock, caused by viruses, rickettsiae,bacterium, spirochaeta, protozoa, nematoda, or the like. This damagecauses enormous losses at home and abroad, and threat of emerging andre-emerging zoonotic diseases by ticks is becoming a serious problem.

Under these circumstances, various methods to exterminate ticks are usedin many countries. Among these methods, the major one is the use ofagents such as organic phosphorus agents, carbamate agents, pyrethroidor macrolide antibiotics, or the like. However, in any agent, a drugresistance is established by using the agent successively or heavily,and thus many agents lose their miticidal activity. Further, when usingsuch agents, it is necessary to take side effects to animals intoconsideration. In addition, there is a problem of a remnant agent whichmay threaten the safety of foods and the environment, and people tend toavoid the use of such agents. Furthermore, the use of agents isapproaching limitation, with respect to the enormous development cost,in addition to the effectiveness thereof and an applicable area. Asdescribed above, it is considered difficult to prevent the parasitism ofticks to humans or animals, and the damage caused by ticks-borneinfection in the 21st century, by means of the use of agents.

In hematophagous arthropods including ticks, acquisition of protectiveimmune response against reinfection in a host against a viral orbacterial infection is known and has been confirmed in the laboratorystage [Fujisaki, Nat. Inst. Anim. Hlth. Quart. (Tokyo), 18, 27–38(1978)]. Due to the recent progress in gene recombination techniques,genes encoding protective antigens, enzymes related to metamorphosisspecific to hematophagous arthropods, or the like are being intensivelycloned in many countries, and an attempt to manufacture safe vaccineproteins or chemotherapeutic agents has been made.

However, such an agent in practical use is only that against Boophilusmicroplus, which was developed by Willadesen [Willadesen and Jogejan,Prasitology Today. 15, 258–262(1999)]. There is now a search for avaccine against Ornithodoros moubata, which is widely distributed overSouthern Europe and the African continent and mediates zoonotic diseasessuch as rickettsiosis, filariasis, Q fever, African recurrent fever, orviral encephalitis, and thus the rapid development and practicalapplication of such a vaccine is greatly desired.

Further, although the development and practical application of a vaccineagainst all ticks is greatly desired, such a vaccine, effective againstall ticks, has not been developed. It is considered that the main reasonfor this is that the breeding of ticks is difficult, and that the searchfor candidate antigens relating to protective immunity against parasiteinfestation has not progressed. Under these circumstances, the searchfor major antigens in Ornithodoros moubata, and the development of aneffective recombinant vaccine in which the major antigen or arecombinant protein thereof is used as an antigen, are desired.

From a medical aspect, it is known that galection is overexpressed in aninflammatory tissue or a tumor tissue, and thus galectin is noted as amarker for an inflammation or tumor. Further, it is known that galectinis involved with an apoptosis of T cells or B cells, and plays animportant role in self recognition. An attempt to use galectin or aninhibitor thereagainst as an immunosuppressive agent for autoimmunedisease, an anti-inflammatory agent, or an antimetastatic agent has beenmade, and there are great expectations that they can be used asmedicaments.

DISCLOSURE OF INVENTION

The present inventors have conducted intensive studies into obtaining anovel polypeptide useful as a candidate for a vaccine against ticks,particularly Ornithodoros moubata, and a polynucleotide encoding thepolypeptide and, as a result, found a novel galectin and apolynucleotide encoding the same. Further, the present inventorsinoculated the galectin into mice, to observe the induction of anantibody production, and confirmed that the galectin is useful as a tickvaccine. The present invention is based on the above findings.

The object of the present invention is to provide a novel galectinuseful as a tick vaccine, and a polynucleotide encoding the galectin.

The object can be solved by a polypeptide of the present invention,i.e., (1) a polypeptide consisting of the amino acid sequence of SEQ IDNO: 2;

-   (2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2    and exhibiting a galectin activity;-   (3) a polypeptide exhibiting a galectin activity and comprising an    amino acid sequence in which one or plural amino acids are    substituted, deleted, and/or inserted at one or plural positions in    the amino acid sequence of SEQ ID NO: 2; or-   (4) a polypeptide comprising an amino acid sequence having a 60% or    more homology with the amino acid sequence of SEQ ID NO: 2, and    exhibiting a galectin activity.

The present invention relates to a polynucleotide encoding thepolypeptide.

The present invention relates to a vector comprising the polynucleotide.

The present invention relates to a transformant comprising thepolynucleotide.

The present invention relates to a process for producing thepolypeptide, comprising the step of culturing the transformant.

The present invention relates to a medicament comprising the polypeptideor a fragment thereof, the polynucleotide, or the vector.

The present invention relates to a pharmaceutical composition comprisingthe polypeptide or a fragment thereof, the polynucleotide, or thevector, and a pharmaceutically or veterinary acceptable carrier ordiluent.

The present invention relates to a method for exterminating ticks,comprising administering to a subject in need thereof the polypeptide ora fragment thereof, the polynucleotide, or the vector in an amounteffective therefor.

The present invention relates to a method for treating or preventing atick-borne infection, comprising administering to a subject in needthereof the polypeptide or a fragment thereof, the polynucleotide, orthe vector in an amount effective therefor.

The present invention relates to an antibody or a fragment thereof,which binds to the polypeptide.

The present invention relates to a method for screening a substancecapable of modifying a galectin activity of the polypeptide, comprisingthe steps of:

bringing the polypeptide into contact with a substance to be tested; and

analyzing the galectin activity of the polypeptide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the result of electrophoresis of a recombinant galectinfusion protein.

FIG. 2 shows the result of electrophoresis by immunoblotting using amonoclonal antibody against hemolymph from 4th instar nymphs.

FIG. 3 shows the result of electrophoresis by immunoblotting using aanti-recombinant galectin fusion protein mouse serum.

FIG. 4 shows the result of agglutination test of the recombinantgalectin fusion protein against mouse erythrocytes.

FIG. 5 shows the result of electrophoresis in a galactose-binding testfor the recombinant galectin fusion protein.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinafter.

[1] Polypeptide of the Present Invention

The polypeptides of the present invention includes

-   (1) a polypeptide consisting of the amino acid sequence of SEQ ID    NO: 2;-   (2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2    and exhibiting a galectin activity;-   (3) a polypeptide comprising an amino acid sequence in which one or    plural amino acids are substituted, deleted, and/or inserted at one    or plural positions in the amino acid sequence of SEQ ID NO: 2, and    exhibiting a galectin activity (hereinafter referred to as a    variation functionally equivalent); and-   (4) a polypeptide comprising an amino acid sequence having a 60% or    more homology with the amino acid sequence of SEQ ID NO: 2, and    exhibiting a galectin activity (hereinafter referred to as a    homologous polypeptide).

The term “galectin activity” as used herein means an activity of bindingto the β-galactoside. Whether or not a polypeptide to be tested exhibitsthe galectin activity may be easily confirmed, for example, by a knownmethod for measuring the galectin activity, in which the polypeptide tobe tested is brought into contact with galactose or a derivativethereof, and then the binding between the polypeptide and galactose or aderivative thereof and/or a degree thereof is analyzed. The method isnot particularly limited, but is preferably confirmed by a methoddescribed in Example 12.

More particularly, for example, a polypeptide to be tested is passedthrough an affinity column (for example, a lactosyl Sepharose column)carrying galactose or a derivative thereof, and then whether or not thepolypeptide to be tested is adsorbed by the column is analyzed. When thepolypeptide to be tested is adsorbed by the column, it may be judgedthat the polypeptide to be tested exhibits the galectin activity.Conversely, when the polypeptide to be tested is not adsorbed by thecolumn, it may be judged that the polypeptide to be tested does notexhibit the galectin activity.

The “polypeptide comprising the amino acid sequence of SEQ ID NO: 2 andexhibiting the galectin activity” as the polypeptide of the presentinvention includes, for example, a fusion polypeptide consisting of anamino acid sequence in which an appropriate marker sequence or the likeis added to the N-terminus and/or the C-terminus of the amino acidsequence of SEQ ID NO: 2, and exhibiting the galectin activity; or

a fusion polypeptide of the polypeptide consisting of the amino acidsequence of SEQ ID NO: 2 and a partner for the fusion, and exhibitingthe galectin activity.

As the marker sequence, for example, a sequence for easily carrying outa confirmation of polypeptide expression, a confirmation ofintracellular localization thereof, a purification thereof, or the likemay be used. As the sequence, there may be mentioned, for example, aFLAG tag, a hexa-histidine tag, a hemagglutinin tag, a myc epitope, orthe like.

As the partner for fusion, there may be mentioned, for example, apolypeptide for purification [for example, glutathione S-transferase(GST) or a fragment thereof], a polypeptide for detection [for example,hemagglutinin or β-galactosidase α a peptide (LacZ α), or a fragmentthereof], a polypeptide for expression (for example, a signal sequence),or the like.

In the above fusion polypeptide, an amino acid sequence which can bespecifically digested with a protease such as thrombin or factor Xa maybe optionally inserted between the polypeptide consisting of the aminoacid sequence of SEQ ID NO: 2 and the marker sequence or the partner forfusion.

The variation functionally equivalent of the present invention is notparticularly limited, so long as it is a polypeptide comprising an aminoacid sequence in which one or plural (preferably 1 to 10, morepreferably 1 to 7, most preferably 1 to 5) amino acids in total (forexample, one to several amino acids in total) are deleted, substituted,and/or inserted at one or plural positions in the amino acid sequence ofSEQ ID NO: 2, and exhibiting the galectin activity. Further, an originof the variation functionally equivalent is not limited to Ornithodorosmoubata.

The variation functionally equivalent of the present invention includesnot only Ornithodoros moubata variations of the polypeptide consistingof the amino acid sequence of SEQ ID NO: 2, but also variationsfunctionally equivalent derived from organisms other than Ornithodorosmoubata [for example, Argasids (soft ticks) other than Ornithodorosmoubata or, or Ixodids (hard ticks)]. Further, it includes polypeptidesprepared using polynucleotides obtained by artificially modifying theiramino acid sequences encoded thereby by genetic engineering techniques,on the basis of polynucleotides encoding these native polypeptides(i.e., Ornithodoros moubata variations or variations functionallyequivalent derived from organisms other than Ornithodoros moubata), oron the basis of polynucleotides encoding the amino acid sequence of SEQID NO: 2. The term “variation” as used herein means an individualdifference between the same polypeptides in the same species or adifference between homologous polypeptides in several species.

Ornithodoros moubata variations of the polypeptide consisting of theamino acid sequence of SEQ ID NO: 2, or variations functionallyequivalent derived from organisms other than Ornithodoros moubata may beobtained by those skilled in the art in accordance with the informationof a base sequence (for example, the base sequence of the 24th to 1025thbases in the base sequence of SEQ ID NO: 1) of a polynucleotide encodingthe polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.In this connection, genetic engineering techniques may be generallyperformed in accordance with known methods (for example, Sambrook etal., “Molecular Cloning, A Laboratory Manual”, Cold Spring HarborLaboratory Press, 1989), unless otherwise specified.

For example, an appropriate probe or appropriate primers are designed inaccordance with the information of a base sequence of a polynucleotideencoding the polypeptide consisting of the amino acid sequence of SEQ IDNO: 2. A polymerase chain reaction (PCR) method (Saiki, R. K. et al.,Science, 239, 487–491, 1988) or a hybridization method is carried outusing a sample (for example, total RNA or an mRNA fraction, a cDNAlibrary, or a phage library) derived prepared from an organism [forexample, Argasids (soft ticks) other than Ornithodoros moubata or, orIxodids (hard ticks)] of interest and the primers or the probe to obtaina polynucleotide encoding the polypeptide. A desired polypeptide may beobtained by expressing the resulting polynucleotide in an appropriateexpression system and confirming that the expressed polypeptide exhibitsthe galectin activity by, for example, the method described in Example6.

Further, the polypeptide artificially modified by genetic engineeringtechniques may be obtained by, for example, the following procedure. Agene encoding the polypeptide is obtained by a conventional method suchas site-specific directed mutagenesis (Mark, D. F. et al., Proc. Natl.Acad. Sci. USA, 81, 5662–5666, 1984). A desired polypeptide may beobtained by expressing the resulting polynucleotide in an appropriateexpression system and confirming that the expressed polypeptide exhibitsthe galectin activity by, for example, the method described in Example6.

The homologous polypeptide of the present invention is not particularlylimited, so long as it is a polypeptide having an amino acid sequencehaving a 60% or more homology with the amino acid sequence of SEQ ID NO:2, and exhibiting the galectin activity. The homologous polypeptide ofthe present invention may have an amino acid sequence having preferablya 70% or more homology, more preferably a 80% or more homology, morepreferably a 90% or more homology, more preferably a 95% or morehomology, most preferably a 98% or more homology, with respect to theamino acid sequence of SEQ ID NO: 2. The term “homology” as used hereinmeans a value obtained by a Clustal program (Higgins and Sharp, Gene,73, 237–244, 1988; and Thompson et al., Nucleic Acid Res., 22,4673–4680, 1994) in accordance with a default parameter.

The above-mentioned novel polypeptide of the present invention may bemanufactured by various known methods, for example, known geneticengineering techniques using the polynucleotide of the present inventionwhich encodes the polypeptide of the present invention. Moreparticularly, the polypeptide of the present invention may be preparedby culturing the transformant of the present invention described below(i.e., the transformant comprising the polynucleotide of the presentinvention) under conditions in which the novel polypeptide of thepresent invention can be expressed, and then separating and purifyingthe desired polypeptide from the resulting culture in accordance with aconventional method for a polypeptide separation and purification. Asthe separation and purification method, there may be mentioned, forexample, salting-out with ammonium sulfate, an ion exchange columnchromatography using ion exchange cellulose, a molecular sieve columnchromatography using molecular sieve gel, an affinity columnchromatography using protein A agarose, dialysis, lyophilization, or thelike.

The present invention includes a fragment of the polypeptide of thepresent invention. The fragment of the present invention is useful as anactive ingredient for the medicament of the present invention or as anantigen for preparing the antibody of the present invention.

[2] Polynucleotide of the Present Invention

The polynucleotide of the present invention is not particularly limited,so long as it encodes the polypeptide of the present invention. As thepolynucleotide of the present invention, there may be mentioned, forexample, a polynucleotide consisting of the 24th to 1025th bases in thebase sequence of the SEQ ID NO: 1. In this connection, the term“polynucleotide” as used herein includes both DNA and RNA.

The present invention includes a polynucleotide comprising a basesequence which can hybridize with the polynucleotide of the presentinvention, preferably a polynucleotide consisting of a base sequencewhich can hybridize with the polynucleotide of the present invention.The base sequence capable of hybridizing with the polynucleotide of thepresent invention is preferably a base sequence complementary to thebase sequence (or a partial sequence thereof) of the polynucleotide ofthe present invention, more preferably a base sequence complementary tothe base sequence (or a partial sequence thereof) consisting of the 24thto 1025th bases in the base sequence of the SEQ ID NO: 1.

[3] Vector and Transformant of the Present Invention

The vector of the present invention is not particularly limited, so longas it comprises the polynucleotide of the present invention. As thevector, there may be mentioned, for example, a vector obtained byintroducing the polynucleotide of the present invention into a knownexpression vector appropriately selected in accordance with a host cellto be used.

The transformant of the present invention is not particularly limited,so long as it comprises the polynucleotide of the present invention. Thetransformant of the present invention may be, for example, a cell inwhich the polynucleotide is integrated into a chromosome of a host cell,or a transformant containing the polynucleotide as a vector comprisingpolynucleotide. Further, the transformant of the present invention maybe a transformant expressing the polypeptide of the present invention,or a transformant not expressing the polypeptide of the presentinvention. The transformant of the present invention may be obtained by,for example, transfecting a desired host cell with the vector of thepresent invention or the polynucleotide of the present invention per se.

The host cell may be, for example, a known microorganism usually used,for example, an Escherichia coli or yeast (Saccharomyces cerevisiae), ora known cultivated cell, such as an animal cell, such as a CHO cell, anHEK-293 cell, or a COS cell, or an insect cell such as a BmN4 cell.

The known expression vector may be, for example, pUC, pTV, pGEX, pKK, orpTrcHis for an Escherichia coli; pEMBLY or pYES2 for the yeast; pcDNA3or pMAMneo for the CHO cell; pcDNA3 for the HEK-293 cell; pcDNA3 for theCOS cell; a vector (such as pBK283) containing a polyhedrin promoter ofa silkworm nucleopolyhederovirus (BmNPV) for the BmN4 cell. Further, theexpression vector includes a virus vector which can be used as a vectorfor a gene therapy, such as retrovirus, adenovirus, or Sendai virus.

[4] Medicament of the Present Invention

The medicament of the present invention (preferably a tick vaccine)comprises, as a active ingredient, the polypeptide of the presentinvention or a fragment thereof, the polynucleotide of the presentinvention, or the vector of the present invention. In the presentinvention, the polypeptide of the present invention or a fragmentthereof, the polynucleotide of the present invention, or the vector ofthe present invention can be orally or parenterally administered alone,or preferably together with a pharmaceutically or veterinarilyacceptable carrier or diluent, to an animal (preferably a mammal,particularly a human) in need of an extermination of ticks.

When the active ingredient in the medicament of the present invention(i.e., the polypeptide of the present invention or a fragment thereof,the polynucleotide of the present invention, or the vector of thepresent invention) is administered to an animal as a tick vaccine, anantibody production may be induced and then ticks may be terminated bydevelopment of protective immunity against reinfection in the hostanimal. Further, as a result, it is possible to treat or preventtick-borne infections such as rickettsiosis, filariasis, Q fever,African recurrent fever, or viral encephalitis.

In other words, the pharmaceutical composition (preferablypharmaceutical composition for exterminating ticks or pharmaceuticalcomposition for treating or preventing a tick-borne infection) of thepresent invention comprises the polypeptide of the present invention ora fragment thereof, the polynucleotide of the present invention, or thevector of the present invention as the active ingredient, and apharmaceutically or veterinary acceptable carrier or diluent. The activeingredient in the present invention (i.e., the polypeptide of thepresent invention or a fragment thereof, the polynucleotide of thepresent invention, or the vector of the present invention) can be usedin the manufacture of the above medicament (preferably medicament forexterminating ticks or medicament for treating or preventing atick-borne infection).

When the medicament of the present invention is used as a tick vaccine,the fragment of the polypeptide of the present invention is notparticularly limited, so long as the fragment administered to a subjectcan induce immunity thereagainst. The fragment can be appropriatelyselected by those skilled in the art.

The medicament (particularly the tick vaccine) of the present inventioncan be used, for example, by mixing the polypeptide of the presentinvention with an adjuvant or the like and inoculating the resultingmixture into an animal (for example, livestock) at an appropriateinterval as a tick vaccine. Further, it can be used by dissolving orsuspending the polypeptide of the present invention directly in anappropriate solvent, or by enclosing it in liposomes or integrating aDNA encoding it in an appropriate vector. Furthermore, it can be used inan appropriate formulation such as injections, tablets, capsules, eyedrops, creams, suppositories, sprays, poultices, or the like, optionallyby adding a pharmaceutical acceptable carrier to the polypeptide of thepresent invention.

As the pharmaceutical acceptable carrier, well-known solvents, bases,stabilizing agents, antiseptics, solubilizing agents, fillers, buffers,and the like may be used. When the polypeptide of the present inventioncontained in the medicament of the present invention is used in theabove formulation, the administration method and the dose may bedetermined in accordance with, for example, the age or sex of eachsubject, or the kind or degree of each disease.

The oral administration includes a sublingual administration. As theparenteral administration, for example, inhalation, percutaneousadministration, ophthalmic administration, vaginal administration,intra-articular administration, rectal administration, intra-arterialadministration, intravenous administration, local administration,intramuscular administration, subcutaneous administration,intraperitoneal administration, or the like may be selected.

It is known that galection is overexpressed in an inflammatory tissue ora tumor tissue, and that galectin is involved with apoptosis of T cellsor B cells and plays an important role in self recognition [H. Leffler,Trends in Glycoscience and Glycotechnology, 6, 9–19(1997)]. Therefore,the polypeptide of the present invention, the polynucleotide of thepresent invention, the vector of the present invention, the antibody ora fragment thereof of the present invention, or a substance capable ofmodifying (for example, suppressing or promoting) the galectin activity,which can be obtained by the screening method of the present invention,is useful as an active ingredient for an immunosuppressive agent forautoimmune disease, an anti-inflammatory agent, an antimetastatic agent,or an agent for inducing or suppressing apoptosis.

The present invention includes an immunosuppressive agent for autoimmunedisease, an anti-inflammatory agent, an antimetastatic agent, or anagent for inducing or suppressing apoptosis comprising, as an activeingredient, the polypeptide of the present invention, the polynucleotideof the present invention, the vector of the present invention, theantibody or a fragment thereof of the present invention, or a substancecapable of modifying (for example, suppressing or promoting) thegalectin activity, which can be obtained by the screening method of thepresent invention.

[5] Antibody and the Fragment Thereof of the Present Invention

An antibody, such as a polyclonal antibody or a monoclonal antibody,which reacts with the polypeptide of the present invention may beobtained by directly administering the polypeptide of the presentinvention or a fragment thereof to various animals. Alternatively, itmay be obtained by a DNA vaccine method (Raz, E. et al., Proc. Natl.Acad. Sci. USA, 91, 9519–9523, 1994; or Donnelly, J. J. et al., J.Infect. Dis., 173, 314–320, 1996), using a plasmid into which apolynucleotide encoding the polypeptide of the present invention isinserted.

The polyclonal antibody may be produced from a serum or eggs of ananimal such as a rabbit, a rat, a goat, or a chicken, in which theanimal is immunized and sensitized by the polypeptide of the presentinvention or a fragment thereof emulsified in an appropriate adjuvant(for example, Freund's complete adjuvant) by intraperitoneal,subcutaneous, or intravenous administration. The polyclonal antibody maybe separated and purified from the resulting serum or eggs in accordancewith conventional methods for polypeptide isolation and purification.Examples of the separation and purification methods include, forexample, centrifugal separation, dialysis, salting-out with ammoniumsulfate, or a chromatographic technique using such as DEAE-cellulose,hydroxyapatite, protein A agarose, and the like.

The monoclonal antibody may be easily produced by those skilled in theart, according to, for example, a cell fusion method of Kohler andMilstein (Kohler, G. and Milstein, C., Nature, 256, 495–497, 1975).

A mouse is immunized intraperitoneally, subcutaneously, or intravenouslyseveral times at an interval of a few weeks by a repeated inoculation ofemulsions in which the polypeptide of the present invention or afragment thereof is emulsified into a suitable adjuvant such as Freund'scomplete adjuvant. Spleen cells are removed after the finalimmunization, and then fused with myeloma cells to prepare hybridomas.

As a myeloma cell for obtaining a hybridoma, a myeloma cell having amarker such as a deficiency in hypoxanthine-guaninephosphoribosyltransferase or thymidine kinase (for example, mousemyeloma cell line P3X63Ag8.U1) may be used. As a fusing agent,polyethylene glycol may be used. As a medium for preparation ofhybridomas, for example, a commonly used medium such as an Eagle'sminimum essential medium, a Dulbecco's modified minimum essentialmedium, or an RPMI-1640 medium may be used by adding properly 10 to 30%of a fetal bovine serum. The fused strains may be selected by a HATselection method. A culture supernatant of the hybridomas is screened bya well-known method such as an ELISA method or an immunohistologicalmethod, to select hybridoma clones secreting the antibody of interest.The monoclonality of the selected hybridoma is guaranteed by repeatingsubcloning by a limiting dilution method. Antibodies in an amount whichmay be purified are produced by culturing the resulting hybridomas in amedium for 2 to 4 days, or in the peritoneal cavity of apristane-pretreated BALB/c strain mouse for 10 to 20 days.

The resulting monoclonal antibodies in the culture supernatant or theascites may be separated and purified by conventional polypeptideisolation and purification methods. Examples of the separation andpurification methods include, for example, centrifugal separation,dialysis, salting-out with ammonium sulfate, or chromatographictechnique using such as DEAE-cellulose, hydroxyapatite, protein Aagarose, and the like.

Further, the monoclonal antibodies or the antibody fragments containinga part thereof may be produced by inserting the whole or a part of agene encoding the monoclonal antibody into an expression vector andintroducing the resulting expression vector into appropriate host cells(such as E. coli, yeast, or animal cells).

Antibody fragments comprising an active part of the antibody such asF(ab′)₂, Fab, Fab′, or Fv may be obtained by a conventional method, forexample, by digesting the separated and purified antibodies (includingpolyclonal antibodies and monoclonal antibodies) with a protease such aspepsin, papain, and the like, and separating and purifying the resultingfragments by standard polypeptide isolation and purification methods.

Further, an antibody which reacts to the polypeptide of the presentinvention may be obtained in a form of single chain Fv or Fab inaccordance with a method of Clackson et al. or a method of Zebedee etal. (Clackson, T. et al., Nature, 352, 624–628, 1991; or Zebedee, S. etal., Proc. Natl. Acad. Sci. USA, 89, 3175–3179, 1992). Furthermore, ahumanized antibody may be obtained by immunizing a transgenic mouse inwhich mouse antibody genes are substituted with human antibody genes(Lonberg, N. et al., Nature, 368, 856–859, 1994).

[6] Screening Method of the Present Invention

It is possible to determine whether or not a substance to be testedmodifies (for example, suppresses or promotes) the galectin activity ofthe polypeptide according to the present invention, using thepolypeptide of the present invention.

Substances to be tested to which may be applied the screening method ofthe present invention are not particularly limited, but there may bementioned, for example, various known compounds (including peptides)registered in chemical files, compounds obtained by combinatorialchemistry techniques (Terrett, N. K. et al., Tetrahedron, 51, 8135–8137,1995), or random peptides prepared by employing a phage display method(Felici, F. et al., J. Mol. Biol., 222, 301–310, 1991) or the like. Inaddition, culture supernatants of microorganisms, natural componentsderived from plants or marine organisms, or animal tissue extracts maybe used as the test substances for screening. Further, compounds(including peptides) obtained by chemically or biologically modifyingcompounds (including peptides) selected by the screening method of thepresent invention may be used.

The screening method of the present invention may be carried out by amethod similar to the above-mentioned method for confirming the galectinactivity, except that the polypeptide of the present invention,galactose or a derivative thereof, and the test substance are broughtinto contact with each other instead of bringing the test polypeptideinto contact with galactose or a derivative thereof.

Namely, in the screening method of the present invention, it isconfirmed whether or not the test substance modifies the galectinactivity of the polypeptide of the present invention by bringing intocontact the polypeptide of the present invention, galactose or aderivative thereof, and the test substance, and then analyzing whetheror not the polypeptide of the present invention binds to galactose or aderivative thereof (or a degree of the binding) in the presence of thetest substance. When the polypeptide of the present invention does notbind to galactose or a derivative thereof, or the degree of the bindingis decreased, it is possible to confirm that the test substancesuppresses the galectin activity of the polypeptide of the presentinvention. Alternatively, when the degree of the binding between thepolypeptide of the present invention and galactose or a derivativethereof is increased, it is possible to confirm that the test substancepromotes the galectin activity of the polypeptide of the presentinvention.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples. The procedures described inthe following Examples were performed in accordance with varioustechniques commonly used in molecular biology, acarology,arthropodology, immunology, or biochemistry, described in, for example,Sambrook et al., “Molecular Cloning, A Laboratory Manual”, Cold SpringHarbor Laboratory Press, 1989 or similar books. As a software foranalyzing DNA, MacVector™ (Oxford Molecular) was used.

Example 1 Preparation of Hybridoma Producing Anti-Tick HemolymphMonoclonal Antibody

Hemolymph was collected from Ornithodoros moubata 4th instar nymphsafter 6 days from engorgement, and the homogenate thereof was sonicatedto obtain a hemolymph solution. After 200 μL of the hemolymph solution(100 μg as a protein amount) was mixed with 200 μL of a completeFreund's adjuvant (Adjuvant Complete Freund; Difco), the mixture wasintraperitoneally inoculated into a 7-week-old female BALB/c mouse.After 14, 21, 28, 42, and 56 days from the intraperitoneal inoculation,200 μL of the hemolymph solution (100 μg as a protein amount) was mixedwith an incomplete Freund's adjuvant (Adjuvant Incomplete Freund;Difco), and each booster inoculation was carried out. After 74 days fromthe intraperitoneal inoculation, the final inoculation with 200 μL ofthe hemolymph solution (100 μg as a protein amount) was carried out fromtail vein. After 3 days, the spleen was removed from the mouse.

The obtained spleen cells and SP2/0-Ag14 myeloma cells were fused usingpolyethylene glycol. The fused cells were cultured in a GIT mediumcontaining 5% fetal bovine serum (FBS)/5% BriClone (Arch Port Ltd.,Dublin, Ireland)/HAT at 37° C. under conditions of 5% CO₂. Using thesupernatant of each culture, clones producing an antibody were screenedby an indirect fluorescent antibody (IFA) method in which the hemolymphfrom Ornithodoros moubata 4th instar nymphs after 6 days fromengorgement was used as an antigen, 10% sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis [Laemmli et al., Nature, 227,680–685 (1970)], and a western blotting method using immunoblotting[Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350–4354 (1979)]. Thescreening and limiting dilution were repeated until obtaining a singleclone, to obtain hybridoma cells producing a monoclonal antibody againstthe hemolymph from Ornithodoros moubata 4th instar nymphs after 6 daysfrom engorgement.

Example 2 Isolation of Gene n4E12-2 Encoding Novel Tick Galectin

Total RNA was extracted from Ornithodoros moubata 4th instar nymphsafter 6 days from engorgement by an Acid Guanidinium-phenol-chloroformmethod [Chomczynski et al., Anal. Biochem., 162, 156–159 (1987)]. Fromthe resulting total RNA, poly A⁺ RNA was purified using an mRNAisolation kit [Oligotex-dT30 (Super), code W9021B; Takara] in accordancewith a protocol attached to the kit.

The following procedures, i.e., construction of a cDNA library,immunoscreening, and insertion into plasmid of a cDNA clone (in vivoExcision) were performed using commercially available reagent kits(Stratagen) in accordance with protocols attached thereto.

More particularly, cDNA was synthesized using 5 ug of Ornithodorosmoubata mRNA as a template and a cDNA synthesis kit (ZAP-cDNA SynthesisKit, Cat. No. 200401-5; Stratagen). The resulting cDNA was fractionatedby a size fractionation with a Sepharose CL-2B gel column, inserted intoa vector (Uni-ZAP XR Vector, Cat. No. 237211; Stratagen), and packagedusing a packaging reagent (GigapackIII Gold packaging extract;Stratagen). Escherichia coli (E. coli XL1-Blue MRF′ strain) wastransfected with the packaged product to obtain a library containingapproximately 500,000 cDNA clones.

The cDNA library was immunoscreened using the monoclonal antibodyobtained in Example 1 against the hemolymph from Ornithodoros moubata4th instar nymphs after 6 days from engorgement, to obtain threeoverlapping positive clones. These clones were inserted into plasmid(i.e., converted into a pBluescript) by an in vivo Excision method.

Each plasmid containing a cDNA fragment was purified using a plasmidpurification kit (Cat no. 12125; Qiagen), and then PCR was carried outusing a sequencing kit (Dye Primer Cycle Sequencing Kit, Part No.4303153; Perkin Elmer) in accordance with a protocol attached to thekit. Each resulting PCR product was analyzed with a DNA sequencer (ABIPRISM 3100 Genetic Analyzer; Perkin Elmer) to determine a base sequenceof each cDNA fragment.

As a result, it was found that all three clones were derived from asingle gene. The longest clone was used in the following analyses.

The full length of the cDNA was 1094 bp, and the base sequence thereofwas that of SEQ ID NO: 1. It was confirmed that the base sequencecontains an open reading frame consisting of 1002 bp (a base sequenceconsisting of the 24th to 1025th bases in the base sequence of SEQ IDNO: 1). The amino acid sequence of a protein deduced from the openreading frame was the amino acid sequence of SEQ ID NO: 2 consisting of333 amino acid residues, and the deduced molecular weight was 36.6 kDa.

Hereinafter, the gene is referred to as an n4E12-2 gene. The homologysearch of the amino acid sequence deduced from the n4E12-2 gene wascarried out by a BLAST method (Basic local alignment search tool;Altschul, S. F. et al., J. Mol. Biol., 215, 403–410, 1990; obtained fromthe National Center for Biotechnology Information). As a result, it wasconfirmed that the amino acid sequence had a high homology with a knowngalectin protein. For example, it had an approximately 27% homology withmouse prostatic cancer antigen galectin.

Hereinafter, the protein encoded by the n4E12-2 gene is referred to as“galectin”. In this connection, it was confirmed in Examples 11 and 12that the protein was galectin.

Example 3 Construction of Vector for Expressing Tick Galectin FusionProtein

The pBluescript plasmid obtained in Example 2, in which the cDNAfragment containing the Ornithodoros moubata n4E12-2 gene was insertedthereinto, was digested with restriction enzymes EcoRI and XhoI. Theresulting cDNA fragment containing the n4E12-2 gene was inserted betweenthe EcoRI site and the XhoI site of a vector pGEMEX-4T-3 (Promega) forexpression in Escherichia coli, and then recombinant clones in which thegalectin ORF fragment was inserted in the same direction as that ofglutathione S-transferase (GST) in the vector were selected. Arecombinant plasmid pGEMEX-4T-3/n4E12-2 was purified using a plasmidpurification kit (Qiagen).

Example 4 Expression of Tick Galectin Recombinant Protein in Escherichiacoli

Escherichia coli JM109 (DE3) (Promega) was transformed with therecombinant plasmid prepared in Example 3, and then the transformantswere cultured in an LB medium containing ampicillin at 37° C. WhenOD_(600nm) of the culture became 0.3˜0.5, isopropyl-thio-galactoside(IPTG) was added to the culture so that the final concentration became0.01 mmol/L, and then the transformants were further cultured for 4hours.

The expression of tick galectin recombinant protein was confirmed bycarrying out 10% SDS-polyacrylamide gel electrophoresis [Laemmli et al.,Nature, 227, 680–685 (1970)] followed by immunoblotting [Towbin et al.,Proc. Natl. Acad. Sci. USA, 76, 4350–4354 (1979)], and then an amidoblack staining.

As a result, the expression of the recombinant protein having amolecular weight of approximately 63 kDa was observed, and it wasconfirmed that the recombinant protein was a fusion protein of a GSTleader protein (26 kDa) and the tick galectin protein (36.6 kDa) (seeFIG. 1).

Example 5 Purification of Tick Galectin Recombinant Protein andPreparation of Antiserum

The recombinant galectin fusion protein expressed in Escherichia coli bythe method described in Example 4 was purified in accordance with aprotocol attached to a commercially available kit (Bulk GST PurificationModule; Amersham Bioscience). More particularly, Escherichia coliinduced with IPTG was collected by centrifugation. The resulting pelletwas sonicated in a TNE buffer containing lysozyme, and then the wholewas centrifuged at 5000 rpm to obtain the pellet. The resulting pellet(insoluble fraction) was solubilized with Triton X-100, and then thewhole was centrifuged at 5000 rpm to obtain the supernatant. Theresulting supernatant was mixed with a glutathione resin, and then themixture was centrifuged at 5000 rpm. The resulting pellet was elutedwith 16 mmol/L glutathione solution to purify a “soluble fraction”.

The result of electrophoresis of the purified recombinant galectinfusion protein is shown in FIG. 1. In this connection, theelectrophoresis, blotting, and staining were performed in the mannersimilar to that described in Example 4. In FIG. 1, the lane on the leftside is the result of molecular weight markers, lane 1 is the result ofthe insoluble fraction, and lane 2 is the result of the solublefraction. The number “63” at the right side of lane 2 means themolecular weight (63 kDa) of the recombinant galectin fusion protein.

An emulsion was prepared by mixing 50 μL of a solution containing 100 μgof the purified recombinant galectin fusion protein with 50 μL of acomplete Freund's adjuvant (Adjuvant Complete Freund; Difco). Theemulsion was intraperitoneally inoculated into a 6-week-old femaleBALB/c mouse. After 2, 4, 6, and 8 weeks from the intraperitonealinoculation, 100 μg of the recombinant galectin fusion protein was mixedwith 50 uL of Titer Max (Gold; CytRx), and each booster inoculation wascarried out. After 2 weeks from the final inoculation, blood wascollected and the resulting serum was kept at −30° C.

Example 6 Identification of Native (Wild Type) Galectin byImmunoblotting Using Anti-Tick Hemolymph Monoclonal Antibody

In this Example, the wild type galectin protein was identified byimmunoblotting [Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350–4354(1979)] using the monoclonal antibody obtained in Example 1 against thehemolymph from Ornithodoros moubata 4th instar nymphs after 6 days fromengorgement. As samples, the hemolymph from Ornithodoros moubata 4thinstar nymphs after 6 days from engorgement prepared in the mannersimilar to that described in Example 1 and a lysate of a tick fat bodywere used. The lysate was prepared by dissecting ticks in PBS (phosphatebuffer), homogenizing the obtained tracheae containing the fat body inPBS, and sonicating the homogenate while cooling with ice.

The result is shown in lanes 5 and 6 in FIG. 2. In this connection,lanes 1 to 4 shows the result obtained in Example 7.

In FIG. 2, lane 5 is the result of the hemolymph from Ornithodorosmoubata 4th instar nymphs after 6 days from engorgement, and lane 6 isthe result of the fat body lysate. The numbers “63” and “43” at the sideof lane 6 mean the molecular weight (63 kDa) of the recombinant galectinfusion protein and the molecular weight (43 kDa) of the wild typegalectin protein, respectively.

As shown in lanes 5 and 6 in FIG. 2, the specific band of 43 kDa wasdetected in the hemolymph from Ornithodoros moubata 4th instar nymphsafter 6 days from engorgement and the fat body lysate. The reason whythe measured molecular weight of the wild type galectin protein ishigher than the theoretical molecular weight (36.6 kDa) seems to be dueto a difference of a post-translational modification.

Example 7 Confirmation of Reactivity of Anti-Tick Hemolymph MonoclonalAntibody with Respect to Recombinant Galectin Fusion Protein

In this Example, the reactivity of the monoclonal antibody obtained inExample 1 against the hemolymph from Ornithodoros moubata 4th instarnymphs after 6 days from engorgement, with respect to the recombinantgalectin fusion protein was examined by immunoblotting. As samples, thesoluble and insoluble fractions derived from Escherichia colitransformed with plasmid pGEMEX-4T-3/n4E12-2 and expressing therecombinant galectin fusion protein (see Example 5), and soluble andinsoluble fractions derived from Escherichia coli transformed withvector pGEMEX-4T-3 and expressing the GST protein were used.

The result is shown in lanes 1 to 4 in FIG. 2.

In FIG. 2, lanes 1 and 2 are the results of the soluble and insolublefractions derived from Escherichia coli transformed with vectorpGEMEX-4T-3 and expressing the GST protein, respectively, and lanes 3and 4 are the results of the soluble and insoluble fractions derivedfrom Escherichia coli transformed with plasmid pGEMEX-4T-3/n4E12-2 andexpressing the recombinant galectin fusion protein. As shown in lane 4in FIG. 2, it was confirmed that the recombinant galectin fusion protein(approximately 63 kDa) reacted with the monoclonal antibody against thehemolymph from Ornithodoros moubata 4th instar nymphs after 6 days fromengorgement. It was shown from the result that the recombinant galectinfusion protein is one of the candidates for a tick vaccine. In thisconnection, the normal mouse serum did not react with the recombinantgalectin fusion protein.

Example 8 Identification of Native (Wild Type) Galectin byImmunoblotting Using Anti-Recombinant Galectin Fusion Protein MouseSerum

In this Example, the wild type galectin protein was identified byimmunoblotting [Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350–4354(1979)] using the anti-recombinant galectin fusion protein mouse serumobtained in Example 5. As samples, the hemolymph from Ornithodorosmoubata 4th instar nymphs after 6 days from engorgement and the fat bodylysate, which were the same as those used in Example 6, were used.

The result is shown in lanes 2 and 3 in FIG. 3. Lanes 1 and 4 are theresults obtained in Example 9.

In FIG. 3, lane 2 is the result of the hemolymph from Ornithodorosmoubata 4th instar nymphs after 6 days from engorgement, and lane 3 isthe result of the fat body lysate. The numbers “63” and “37” at the sideof lane 4 mean the molecular weight (63 kDa) of the recombinant galectinfusion protein and the molecular weight (37 kDa) of the wild typegalectin protein, respectively.

As shown in lanes 2 and 3 in FIG. 3, two specific bands of 37 kDa and 43kDa were detected in the hemolymph from Ornithodoros moubata 4th instarnymphs after 6 days from engorgement and the fat body lysate. The 37 kDaband agreed with the theoretical value (36.6 kDa) of the galectinprotein. In this connection, the reason why the 43 kDa band was detectedbut the 37 kDa band was not detected in Example 6 seems to be due to asmall amount of sample used in electrophoresis.

Example 9 Confirmation of Reactivity of Anti-Recombinant Galectin FusionProtein Mouse Serum with Respect to Recombinant Galectin Fusion Protein

In this Example, the reactivity of the anti-recombinant galectin fusionprotein mouse serum obtained in Example 5 with respect to therecombinant galectin fusion protein was examined by immunoblotting. Assamples, the purified recombinant galectin fusion protein derived fromEscherichia coli transformed with plasmid pGEMEX-4T-3/n4E12-2 andexpressing the recombinant galectin fusion protein (see Example 5), andthe insoluble fraction (before purification) derived from Escherichiacoli transformed with vector pGEMEX-4T-3 and expressing the GST proteinwere used.

The result is shown in lanes 1 and 4 in FIG. 3.

In FIG. 3, lane 1 is the result of the purified recombinant galectinfusion protein, and lane 4 is the result of the (insoluble) GST proteinbefore purification.

As shown in lane 1 in FIG. 3, it was confirmed that the recombinantgalectin fusion protein (approximately 63 kDa) reacted with theanti-recombinant galectin fusion protein mouse serum. It was shown fromthe result that the recombinant galectin fusion protein is one ofcandidates for a tick vaccine. In this connection, the normal mouseserum did not react with the recombinant galectin fusion protein.

Example 10 Confirmation of Antigenicity of Recombinant Galectin FusionProtein Against Mouse

In this Example, whether or not the recombinant galectin fusion proteinprepared in Example 5 can induce an antibody production in a mouse wasexamined.

From 11 mice immunized with the recombinant galectin fusion protein inExample 5, 11 mouse antisera were obtained. Each mouse antiserum wasreacted with a membrane onto which the purified recombinant galectinfusion protein had been previously transferred, and then an antibodywhich bound to the antigen was detected by a western blotting method. Inthis connection, two mouse antisera prepared from two mice immunizedwith an adjuvant were used as a control.

As a result, an antibody against the recombinant galectin fusion proteinwas detected in all antisera derived from mice immunized with therecombinant galectin fusion protein, whereas no antibody was detected inthe control mouse antisera.

From the result, it was confirmed that production of an antibody againstthe recombinant galectin fusion protein was induced by immunizing thefusion protein to a mouse, and that the recombinant galectin fusionprotein is useful as an anti-tick vaccine.

Example 11 Confirmation of Agglutinin Activity of Recombinant GalectinFusion Protein to Mouse Erythrocytes

In this Example, the agglutination of the recombinant galectin fusionprotein to mouse erythrocytes.

More particularly, after mouse erythrocytes were washed with PBS, thepurified recombinant galectin fusion protein was diluted and added tothe erythrocytes, to determine the effective concentration thereof. Onthe basis of the determined effective concentration, lactose, which isan inhibitor of galectin, was added so that the final concentrationbecame 10 mmol/L. Further, using the anti-galectin mouse serum preparedin Example 5, an effect of the serum to suppress agglutination bygalectin was examined.

The result is shown in FIG. 4. In FIG. 4, wells 1 to 4 are the resultswhen 5 μg, 0.25 μg, 0.025 μg, and 0.0025 μg of the recombinant galectinfusion protein were added, respectively, well 5 is the result obtainedwhen 0.25 μg of the recombinant galectin fusion protein and 10 mmol/Llactose were added, and well 6 is the result obtained when 0.25 μg ofthe recombinant galectin fusion protein and 10 μL of the anti-galectinmouse serum prepared in Example 5 were added.

As shown in FIG. 4, the recombinant galectin fusion protein agglutinatedmouse erythrocytes dose-dependently. Further, the reaction was inhibitedby the anti-recombinant galectin fusion protein mouse serum prepared inExample 5 or 10 mmol/L lactose.

It was confirmed from the results that the recombinant galectin fusionprotein exhibits a lectin activity.

Example 12 Confirmation of Galactose-Binding Activity in RecombinantGalectin Fusion Protein

In this Example, a galactose-binding activity in the recombinantgalectin fusion protein was examined by a batch method using lactosylSepharose.

More particularly, the purified recombinant galectin fusion protein andlactosyl Sepharose were mixed in a 1.5 mL-microtube, and centrifuged at5000 rpm. The resulting pellet was washed with PBS-T three times, andeluted with 100 mmol/L lactose.

The result is shown in FIG. 5. In FIG. 5, lane 1 is the result obtainedwhen the recombinant galectin fusion protein before mixing with lactosylSepharose (hereinafter referred to as a preapplied sample) waselectrophoresed, lane 2 is the result obtained when the supernatantobtained by centrifuging the suspended mixture of the recombinantgalectin fusion protein and lactosyl Sepharose was electrophoresed,lanes 3 to 5 are the results when the first to third wash liquids wereelectrophoresed, respectively, and lane 6 is the result obtained whenthe supernatant after elution with lactose. The number “63” at the sideof lane 6 means the molecular weight (63 kDa) of the recombinantgalectin fusion protein.

As shown in FIG. 5, galectin was eluted with lactose (lane 6), and theamount of galectin was almost equal to that of the recombinant galectinfusion protein contained in the preapplied sample (lane 1). Further, therecombinant galectin fusion protein exhibited a property of binding tolactosyl Sepharose, and was eluted with 100 mmol/L lactose.

It was shown from the results that the recombinant galectin fusionprotein exhibits a galactose-binding activity.

INDUSTRIAL APPLICABILITY

According to the polypeptide, polynucleotide, vector, transformant, andantibody of the present invention, the medicament of the presentinvention, particularly a tick vaccine, can be provided.

Further, according to the medicament of the present invention,particularly a tick vaccine, it is possible, for example, to exterminateticks, or to treat or prevent tick-borne infections such asrickettsiosis, filariasis, Q fever, African recurrent fever, or viralencephalitis.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

The invention claimed is:
 1. A substantially pure polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2 or a polypeptide having 90% ormore identity with the amino acid sequence of SEQ ID NO: 2 and havinggalectin activity.
 2. An isolated polynucleotide encoding thepolypeptide according to claim
 1. 3. An expression vector comprising thepolynucleotide according to claim
 2. 4. A host cell comprising theexpression vector according to claim
 3. 5. A method for producing apolypeptide comprising the amino acid sequence of SEQ ID NO: 2 or apolypeptide having 90% or more identity with the amino acid sequence ofSEQ ID NO: 2 and having galectin activity, comprising culturing the hostcell according to claim 4 under conditions promoting expression of thepolypeptide, and recovering the polypeptide from the cell culture.
 6. Apharmaceutical composition comprising the polypeptide according to claim1, and a pharmaceutically or veterinary acceptable carrier or diluent.7. A method for determining whether a test compound is a suppressor ofgalectin activity, said method comprising: (a) determining the galectinactivity of the polypeptide of claim 1 in the presence of a testcompound, and (b) comparing the galectin activity determined in (a) withthe galectin activity determined for the polypeptide of claim 1 in theabsence of the test compound, wherein when said galectin activity of thepolypeptide of claim 1 is lower in the presence of said test compoundthan in the absence of said test compound, said test compound isdetermined to be a suppressor of galectin activity.
 8. A method fordetermining whether a test compound is a promoter of galectin activity,said method comprising: (a) determining the galectin activity of thepolypeptide of claim 1 in the presence of a test compound, and (b)comparing the galectin activity determined in (a) with the galectinactivity determined for the polypeptide of claim 1 in the absence of thetest compound, wherein when said galectin activity of the polypeptide ofclaim 1 is higher in the presence of said test compound than in theabsence of said test compound, said test compound is determined to be apromoter of galectin activity.
 9. A substantially pure polypeptidecomprising the amino acid sequence of SEQ ID NO:2.
 10. A substantiallypure polypeptide consisting of the amino acid sequence of SEQ ID NO:2.